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目的采用高效液相色谱法(high performance liquid chromatography,HPLC)测定钩端螺旋体疫苗中的苯酚含量。方法色谱条件:色谱柱:Novo-Pak C-18层析柱(3.9 mm×150 mm,4μm),流动相:甲醇-超纯水(40∶60,v∶v),流速:1.0 ml/min,检测波长:270 nm,柱温:30℃,进样量:10μl。对建立的方法进行线性范围、精密性、重现性、准确性验证及初步应用。结果苯酚浓度在18.552~129.864μg/ml范围内,色谱峰面积与苯酚浓度呈良好的线性关系(r=0.999 7);苯酚对照品溶液连续进样6次,主峰面积的相对标准偏差(RSD)为0.25%,保留时间的RSD为0.10%;用建立的方法检测6份钩端螺旋体疫苗样品中苯酚含量的RSD为0.5%;准确性试验的总平均回收率为100.6%,RSD为1.0%;HPLC法和溴量滴定法测定4批钩端螺旋体疫苗样品中苯酚含量的结果基本一致。结论 HPLC法可以更准确地测定苯酚含量,且该方法操作简便、快速,精密性高,重现性好,无干扰,适用于钩端螺旋体疫苗成品中苯酚含量的检测。
Objective To determine the phenol content of Leptospira vaccine by high performance liquid chromatography (HPLC). Methods Chromatographic conditions: Novo-Pak C-18 column (3.9 mm × 150 mm, 4 μm), mobile phase: methanol-ultrapure water (40:60, v:v), flow rate: 1.0 ml / min , Detection wavelength: 270 nm, column temperature: 30 ℃, injection volume: 10μl. The established method of linear range, precision, reproducibility, accuracy verification and preliminary application. Results The phenol concentration ranged from 18.552 to 129.864μg / ml. The peak area of phenol showed a good linear relationship with phenol concentration (r = 0.999 7). The phenol standard solution was continuously injected six times and the relative standard deviation (RSD) RSD of retention time was 0.10%; RSD of phenol content in 6 samples of Leptospira was detected by the established method; 0.5%; the average recovery of accuracy test was 100.6% with RSD of 1.0% The results of HPLC and bromometric titration of phenol content in four samples of Leptospira vaccine were basically consistent. Conclusion The HPLC method can determine phenol content more accurately. The method is simple, rapid, accurate, reproducible and non-interfering and can be applied to the detection of phenol in the finished product of Leptospira vaccine.